Innate type 2 lymphocytes (ILC2, also called natural helper cells or nuocytes) are critically involved in widespread infectious and allergic diseases. By residing at mucosal barriers and responding to epithelial-derived cytokines such as IL-33 and IL-25, they rapidly release the early type 2 cytokines that promote the Th2 inflammatory responses of adaptive CD4 T cells and the IgE production of B cells. However, the definition of the ILC2 lineage remains tentative and somewhat controversial in the absence of molecular signature. Furthermore, while they derive from the same common lymphoid precursors (CLP) as other adaptive and innate lymphocytes, their developmental intermediates and their relationship with other related lineages are unknown. This project builds on preliminary studies of PLZF-IRES-GFPCre reporter mice produced in our laboratory showing that the transcription factor PLZF, previously identified as the signature of the innate-like IL- 4/IL-13-producing NKT cell lineage, is also expressed at high levels during the development of ILC2s. The central hypothesis is that PLZF exerts critical functions in the development of ILC2s and that PLZF- reporter/deleter mice can be used to genetically track and manipulate ILC2 precursors and their mature progeny. The objective of this application is to use PLZF-IRES-GFPCre mice to identify the bone marrow precursors of ILC2, characterize their molecular program and define their progeny through fate mapping after crossing to ROSA26-FL-STOP-FL-Tomato mice. Furthermore, the project will test various approaches to genetically manipulate or ablate ILC2 in vivo as a prelude to characterizing their function in physiology and in allergic airway inflammation. The specific aims are: 1) to characterize the PLZFhi subset of CLP; 2) to determine the role of PLZF in ILC2 development and function; 3) to create ILC2-defective mouse models for studies of allergic airway inflammation. The project is innovative because it explores the novel idea that PLZF expression is shared across innate and innate-like lineages specializing in type 2 responses and because it will produce new models and reagents for studies of ILC2s in vivo. This would signal a major shift in our ability to understand and further manipulate the role of innate immunity in worldwide diseases caused or cured by type 2 immunity. The proposed research is significant because it will considerably enhance our understanding of innate lymphocyte lineage development. The concepts and tools developed will make it possible to develop agents that target distinct components of innate immunity to parasites and allergens.